Preparation of a seasoning agent

ABSTRACT

A SEASONING AGENT HAVING A MEAT-LIKE FLAVOR IS PRODUCED BY THE STEPS OF SUBJECTING YEAST TO DECOMPOSITION BY CELL LITIC ENZYMES PRRODUCED BY ORGANISMS OF THE GENERA CORPRINUS, DAEDALEOPSIS OR IRPEX TO PRODUCE A DECOMPOSITION LIQUOR, FILTERING THE DECOMPOSITION LIQUOR TO PRODUCE A FILTRATE, ADJUSTING THE PH OF THE FILTRATE TO 5 TO 6.5 AND HEATING THE FILTRATE AT 40 TO 85*C. TO PRODUCE THE SEASONING AGENT.

United States Patent US. Cl. 426-60 7 Claims ABSTRACT OF THE DISCLOSUREA seasoning agent having a meat-like flavor is produced by the steps ofsubjecting yeast to decomposition by cell lytic enzymes produced byorganisms of the genera Coprinus, Daedaleopsis or Irpex to produce adecomposition liquor, filtering the decomposition liquor to produce afiltrate, adjusting the pH of the filtrate to to 6.5 and heating thefiltrate at 40 to 85 C. to produce the seasoning agent.

This is a continuation of application Ser. No. 4,107, filed Jan. 19,1970, now abandoned.

BACKGROUND OF THE INVENTION This invention relates to a method forpreparing a yeast extract having a harmonizedtaste and flavor byallowing a novel cell lytic enzyme to react with a yeast.

Recently, meat extracts, yeast extracts, etc. which can develop naturaltaste and flavor, have been widely utilized in place of chemicalseasoning agents such as sodium glutamate, sodium 5-inosinate, sodium5-guanylate, sodium succinate, etc., which lack natural flavors andtastes.

Yeast extract has previously been prepared from cultured yeast as a rawmaterial by (1) an extraction method, (2) a protease decompositionmethod, (3) an acid decomposition method, and (4) an autolysis method,but none of these conventional methods is completely satisfactorybecause each has a number of deficiencies. For example, in methods (1)and (2), the yield is low and a bitter taste develops; in method (3) anundesirable smell of acid decomposition is generated and usefulcomponents may be broken down; and in method (4) the control of theprocess is diflicult and the method cannot be applied to heat-driedyeast cells.

From the stand-point of improving the status of such conventionalmethods, the present inventors have made various studies of eliminatingthese deficiencies and preparing a yeast extract of good quality in ahigh yield, and as a result, have found that yeast extract which cansufficiently meet the desired object can be obtained by allowing a novelcell lytic enzyme previously discovered by the present inventors anddescribed in Japanese patent application No. 44608/68 (U.S. applicationSer. No. 828,316 filed May 27, 1969) to react with yeasts or materialcontaining yeasts.

The present invention has been accomplished on the basis of saidfinding, and an object of the present invention is to obtain a yeastextract having a harmonized taste and flavor by allowing a strong celllytic enzyme, which is formed by strains of organisms belonging to thegenera Coprinus, Daedaleopsis, and Irpex, and which has strongglucanase, and protease activities and the ability characteristically todecompose and solubilize yeast cells in a short period of time, to reactwith yeast cells or materials containing yeast cells, therebydecomposing the cell walls as well as decomposing and solubilizingprotein in the cells thereby to form sugars, amino acids, peptides,etc.,

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and then concentrating or pulverizing the solubilized components.

The cell lytic enzymes used in the present invention can be obtained byculturing in a solid medium or a liquid medium strains belonging to thegenus Coprinus, such as Coprinus macrorhizus f. microsporus, Caprinusradians, Coprinus micaceus, etc. or strains belonging to the genusDaedaleopsis, such as Daedaleopsis styracina, Daedaleopsis confragosa,Daedaleopsis tricolor, Daedaleopsis nipponica, etc., or further Irpexlacteus belonging to the genus Irpex. It is necessary that a carbonsource, a nitrogen source and other necessary components for growth ofsaid microorganisms, which can be utilized by said microorganisms, arecontained in the medium. That is, as a carbon source, sucrose, starch,lactose, maltose, dextrose, etc. can be used. As a nitrogen source,distillers solubles, peptone, meat extract, yeast, soybean cake, gluten,amino acids, ammonium salts, nitrates, urea, etc. can be used. Inaddition, magnesium salts, potassium salts, sodium salts, phosphates,etc. or zinc, manganese, iron, cobalt, etc. as trace elements, can beused, if desired.

The cell lytic enzyme is generally prepared from an enzyme solutionobtained by removing cells from a termentation liquor according toconventional enzyme purification methods such as organic solventprecipitation, salting out, concentration under reduced pressure,adsorption and desorption by ion exchange. However, the cells, culturefiltrate, etc. after the culturing in said medium, can also be used asan enzyme source without further treatment.

The raw material yeast used in the present invention includes allavailable yeasts such as bakers yeast cultured according to conventionalmethods, sulfite pulp yeast cultured in a sulfite pulp Waste liquor, theso-called petroleum yeast cultured in a hydrocarbon medium such asnormal paraffin, alcohol waste liquor yeast separated from the alcoholfermentation waste liquor, etc. These yeasts can be used in a driedstate after culturing and separation, or in the form of compressedyeast, or the culture liquor can be employed without further treatment.The decomposition efiiciency of the cell lytic enzyme is generallyincreased when cells killed by heat treatment are used, but the livingcells can also be used without any problem.

The yeast concentration, amount of enzyme added, reaction temperature,reaction time, reaction pH, etc. for preparing the yeast extract dependupon the kind of enzyme used and a sampling method of an enzyme source.Thus, it is necessary to select those which meet the conditions for eachcase. For example, in the case where the enzyme obtained from thestrains belonging to the genus Coprinus such as Coprinus macrorhizus f.microsporus, Coprz'nus radians, etc. is used, the optimum conditionsinclude use of a suspension of cells whose yeast concentration is 5-25pH adjusted to 8-8.5 with caustic soda and sodium secondary phosphate,0.02-1.5% of enzyme added thereto on the basis of dry raw materialyeast, and decomposition carried out at 50-55 C. for 5- 48 hours withstirring. When the enzyme obtained from the strains belonging to thegenus Daedaleopsis or Irpex such as Daedaleopsis styracina, Irpexlacteus, etc. is used, it is preferred that 0.02-2% of enzyme is addedto the suspension of yeast cells at a 5-25% concentration and enzymaticdecomposition is carried out at a pH of 2.5- 3.0 and 45-50 C. for 5-48hours.

These decomposition reactions are not generally troubled byputrefaction, because the temperature is generally high, but ethylacetate, alcohols, etc. can be added and used as preservatives, ifnecessary.

By the decomposition reaction, the cell walls of yeast cellspolysaccharides and proteins are decomposed, and such sugars as glucose,mannose, galactose, ribose, etc.,

various palatable amino acids such as glutamic acid and alanineprincipally, and lower and medium peptides are thereby formed. On theother hand, various vitamins, amino acids, nucleic acid-relatedsubstances, organic acids and phosphoric acid compounds contained in theyeast cells or culture liquor are utilized as soluble components. Whenthe yeast is decomposed by Caprinus macrorhizus f. microsporus, morethan 90% of the yeast cells is solubilized and recovered as yeastextract. Further, in .the decomposition products, no bitter-tastingsubstance is formed. It is possible to increase the amount of aminoacids formed, by adding a commercially available protease preparationthereto after the decomposition by said cell lytic enzyme.

The thus-obtained decomposition liquor is filtered and the filtrate maybe adjusted to the desired taste and flavor by treatment with activatedcarbon, ion exchange resin or nonioic exchange resin. n the other hand,the taste and flavor of the decomposition liquor can be also adjusted byadding various amino acids and organic acids such as nucleotides andsodium glutamate thereto.

Then, the pH of the decomposition filtrate is adjusted to 5-6.5 andreaction is carried out by heating the filtrate at 40-85 C. to promotereaction between the sugars and amino acids formed by decomposition,whereby aging is eifected. The seasoning liquor which has been aged isused as yeast extract as is, or a paste of yeast extract is obtained byconcentration or a powdered yeast extract is prepared by spray drying.

As explained above, by using the novel cell lytic enzyme, the presentinvention permits a number of simultaneous advantages, and thereforeprovides a very useful method. For example, yeast extract can beprepared in high yield from the yeast; good seasoning agents can beobtained from dry yeasts such as petroleum fermentation yeast, sulfitepulp yeast etc.; no substances having bitter taste are formed during thedecomposition; and the salt content of the yeast extract is low.

The present invention is hereunder explained in detail, referring tospecific examples:

EXAMPLE 1 Coprz'nus macrorhizus f. microsporus (ATCC 20120) was culturedunder aeration with stirring at 28 C. for 48 hours in 20 l. or a mediumconsisting of 3% sucrose, 3% of distillers solubles, 0.5% of yeastcells, 0.02% of magnesium sulfate and 0.5 of potassium primary phosphate(pH 6.0), the cells were filtered oif, and a filtrate was obtained.Precipitates obtained by adding acetone to the filtrate to give 75%acetone (volume/volume) were freeze-dried, whereby 170 g. of crudeenzyme was obtained.

In a separate vessel, 80 l. of a suspension of sulfite pulp waste liquoryeast, Candida utilis, having a concentration and a pH adjusted to 8.5was heat-treated at 100 C. for 10 minutes, and then cooled and adjustedto 55 C. A quantity of 17 g. of crude cell lytic enzyme was addedthereto and decomposition was conducted at 55 C. with stirring for 10hours. The cells were decomposed in that decomposition step, and sugars,amino acids, peptides, etc., were liberated. After the decompositionliquor was adjusted to a pH of 6.0 with hydrochloric acid, 62 l. ofseparated liquor was obtained by centrifugal separation. Total solidcontent of the separated liquor was 118 g./l., and thus more than 90% ofthe raw material yeast was deemed to be solubilized. The liquor washeated at 60 C. for 2 hours, then concentrated to give 8.8 kg. of yeastextract in a paste state. General analytical values of the thus obtainedseasoning agent were 7.9% total nitrogen, 4.4% sodium glutamate, 8.2%total sugars, 14.3% ash, 2.8% sodium chloride and 16% water.

A palatability test of a 2% aqueous solution of the present yeastextract was conducted by a staff panel of 5 members of Tokyo 'ResearchLaboratory of Kyowa Hakko Kogyo Co., Ltd. according to the profilemethod, and as 4 a result no bitter taste was observed, but a thick,good taste was felt, a meat-like taste and flavor and a good body wererecognized, and other good evaluations were given by all panel members.

EXAMPLE 2 18 l. of filtrate was obtained by filtering a culture liquorof Coprinus macrorhizus f. microsporus cultured in essentially the samemanner as in Example 1.

In a separate vessel, 82 l. of water and 18 l. of said culture filtrateof cell lytic enzyme were added to 10 kg. of dry yeast cells obtained byculturing a yeast belonging to the genus Candida using normal parafiinsas a carbon source and washing them with hexane and water. The pH wasadjusted to 8.5 with caustic soda, and the yeast cells were decomposedat 55 C. with stirring for 8 hours. Then, the pH of the decompositionliquor was adjusted to 6.0 with hydrochloric acid, and l. of separatedliquor was obtained by centrifugal separation. A quantity of 400 g. ofactivated carbon sold under the trademark Taiko-zinc chloride carbon SAby Futamura Kagaku Kogyo Co., Ltd. was added to the liquor and stirredat 60 C. for one hour, and then filtered. An amount of 100 g. of sodium5'- inosinate was added and the filtrate was dried in a spray drier,whereby 10.2 kg. of light yellow powder was obtained.

The general analytical values of the product seasoning agent were 4%Water, 2.5% sodium chloride, 8.1% total nitrogen, 5.1% sodium glutamateand 17.5% ash, and no bitter taste, disagreeable taste or oil smellwhatsoever were observed.

EXAMPLE 3 Daedaleopsis styracz'na (ATCC 20188) was cultured underaeration with stirring for 72 hours in 20 l. of a medium consisting of3% of sucrose, 3% of distillers solubles, 0.3% of yeast extract, 0.04%of magnesium sulfate and 0.5 of potassium primary phosphate (pH 4.5),the cells were filtered otf and subjected to acetone dehydrationaccording to the conventional method, whereby 400 g. of dry cells wereobtained.

In a separate vessel, 50 l. of 8% yeast suspension (dry basis) wasadjusted to pH 3.0, heat-treated at 100 C. for 10 minutes and thencooled. The entire amount of the so-obtained dry cells of Daedaleopsisstyracina was added to the suspension and the decomposition was carriedout at 45 C. for 5 hours to dissolve the yeast cells. The pH was thenadjusted to 7.2 with caustic soda, and then 8 g. of the protease enzymesold under the trademark Prozyme by Kyowa Hakko Kogyo Co., was added anddecomposition was carried out at 48 C. for three hours to form a largequantity of amino acids. Then, the pH was adjusted to 6.0 withhydrochloric acid, and 38 l. of separated liquor was obtaned bycentrifugal separation. The total solids present in the product liquorwas 95 g./l., and of the yeast cells was deemed to be solubilized. Fromthis liquor, 20 l. of a liquid seasoning agent was obtained byconcentrating under reduced pressure.

EXAMPLE 4 lrpex lacteus (ATCC 20123) was cultured under aeration withstirring for 72 hours in 20 l. of a medium consisting of 3% of sucrose,3% of distillers solubles, 0.5% of yeast cells, 0.05% of magnesiumsulfate and 0.6% of potassium primary phosphate (pH 5.0), the cells werefiltered off, and a precipitate was obtained by adding 65% (weight/volume) of ammonium sulfate to the filtrate. The precipitate weredissolved in water and subjected to elec trodialysis, and 19 g. of crudeenzyme was obtained by freeze-drying.

In a separate vessel, a yeast fermentation liquor was obtained byculturing Saccharomyces cerevisiae at 28 C. for 30 hours in 200 l. of amedium consisting of 5% of glucose, 0.5% of peptone, 0.5% of yeastextract, 0.15% of potassium secondary phosphate, 0.02% of magnesiumsulfate and 0.2% of calcium carbonate (pH 6.0). The resuiting cultureliquor was heat-treated at 95 C. for minutes, and then cooled. The pHwas adjusted to 3.0 with hydrochloric acid. The entire amount of thecrude enzyme was added thereto and the cells were decomposed at 50 C.for 10 hours. The pH was then adjusted to 5.8 with caustic soda, and 185l. of separated liquor was obtained by centrifugal separation. Theresulting liquor contained various organic acids and nucleicacid-related substances formed by the yeast together with thedecomposition product of the yeast. An amount of '9 kg. of activatedcarbon sold under the trademark Taiko-zinc chloride carbon SA byFutamura Kagaku Kogyo Co., Ltd. was added to 185 l. of the separatedliquor, stirred at 60 C. for one hour, and then filtered, whereby afiltrate was obtained. A quantity of 2 kg. of sodium glutamate was addedto the filtrate and 27 kg. of a seasoning agent in a paste state wasobtained by concentration under reduced pressure.

EXAMPLE 5 18 l. of filtrate was obtained by filtering a culture liquorof Coprinus radians (ATCC 20014) and Coprinus micaceus (ATCC 20122)cultured in essentially the same manner as in Example 1.

In a separate vessel, 82 l. of water and 18 l. of said culture filtrateof cell lytic enzyme were added to 10 kg. of dry yeast cells obtained byculturing a yeast belonging to the genus Candida using normal paraffinsas a carbon source and washing them with hexane and water. The pH wasadjusted to 8.5 with caustic soda, and the yeast cells were decomposedat 55 C. with stirring for 8 hours. Then, the pH of the decompositionliquor was adjusted to 6.0 with hydrochloric acid, and 80 1. ofseparated liquor was obtained by centrifugal separation.

A quantity of 400 g. of activated carbon sold under the trademarkTaiko-zinc chloride carbon SA by Futamura Kagaku Kogyo Co., Ltd. wasadded to the liquor and stirred at 60 C. for one hour, and thenfiltered. An amount of 100 g. of sodium 5'-inosinate was added and thefiltrate was dried in a spray drier, whereby 9.8 kg. and 10.5 kg. oflight yellow powder was obtained.

What is claimed is:

1. A process for producing a seasoning agent having a meat like flavorwhich comprises decomposing yeast cells with a cell lytic enzymeobtained from culturing a microorganism selected from the groupconsisting of Coprinus macrorhizus f. microsporus, Coprinus radians,Coprinus micaceus, Daedaleopsis styracina and Irpex lacteus in anutrient medium, to obtain a decomposition liquor, filtering saidliquor, adjusting the filtrate to pH 5 to 6.5 and thereafter heatingsaid filtrate at 40 to 85 C. to eifect aging whereby there occursreaction of sugars and amino acids formed by decomposition to producesaid meat-like flavor.

2. Process according to claim 1 wherein said yeast cells are killed byheat treatment prior to said decomposition step.

3. Process according to claim 1 wherein said decomposition liquor istreated with a protease enzyme.

4. Process according to claim 1 wherein said aged filtrate isconcentrated to obtain a paste.

5. Process according to claim 1 wherein said aged filtrate is spraydried to obtain a powder.

6. Process according to claim 1 wherein said decomposition stepcomprises reacting 0.02 to 1.5% of cell lytic enzyme obtained from amicroorganism selected from the group consisting of Coprinus macrorhizusf. microsporus, Co prinus radians and Coprinus micaceus, with a 5 to 15%yeast cell suspension for 5 to 48 hours at a pH of 8 to 8.5 and atemperature of 50 to C.

7. Process according to claim 1 wherein said decomposition stepcomprises reacting 0.02 to 2% of cell lytic enzyme obtained from amicroorganism selected from the group consisting of Daedaleopsisstyracina and Irpex lacteus, with a 5 to 25% yeast cell suspension for 5to 48 hours at a pH of 2.5 to 3.0 and temperature of 45 to 50 C.

References Cited OTHER REFERENCES Ozaki et. al., Effects of the CultureFiltrate of Irpex Lactens on the Extraction rate of Protein fromDefatted soybean. Chemical Astracts, vol. '65, 1966 (pp. 1344 and1345a), QDIASl.

Clements et al., The Genera of Fungi, Hafnet Publ. Co., New York 1954(pp. 157, 163, 164 and 168) QK60306.

DAVID M. NAFF, Primary Examiner US. Cl. X.R.

' UNITED STATES A E T I 0FF1C v I CERTIFICATE OF CORRECTION Patent No.3,309 7 I' I Dated- Inven KENGO ISHIDA. at al It is certified thaterropappears if! the ,abdveidentified patent and that saidLetters'Patent are hereby corrected as shownblow;

Identify as an inventor:

ATSUSHI YAMAMOTO, Tokyo, Japan Signed and sealed this 10th day of Septemb e'r 1974,

(SEAL) Attest: v I MCCOY M. GIBSON, JR. c.'1MARs ALL-; DANN 1 AttestingOfficer Commissioner of Patents;

